The present invention relates to molecular complexes which include local anesthetics, to pharmaceutical compositions comprising the same, and to methods of making and using the same.
Local anaesthetics are a large group of compounds which include several classes of compounds. Physiologically active natural or synthetic chemicals or biologicals and their derivatives act at/in cell membranes and/or specific receptor molecules at/in cell membranes in vivo or in vitro. Local anesthetics are described in De-Paula, E. And S. Schreier (1996) Braz. J. Med. Res. 29:877-894 and Ritchie, J. M. and N. M. Greene xe2x80x9cLocal Anestheticsxe2x80x9d, Chapter 15 302-321, which are both incorporated herein by reference, describe local anesthetics and their uses.
In addition, the use of local anesthetics in combination with nucleic acid molecules for in vivo gene transfer generally and in particular for protective and therapeutic vaccination protocols as well as gene therapy and antisense protocols is described in U.S. Pat. No. 5,593,972 issued on Jan. 14, 1997 to Weiner et al. and in PCT application PCT/US94/00899, which are both incorporated herein by reference. This application claims priority to U.S. Provisional Application No. 60/045,122 filed Apr. 30, 1997, which is incorporated herein by reference.
The present invention relates to compositions that comprise lamellar vesicles that comprise a local anesthetic and a nucleic acid molecule. In some embodiments, the proportion of local anesthetic to nucleic acid in the vesicles is 0.01-2.5% weight to volume (w/v) local anesthetic to 1 xcexcg/ml-10 mg/ml nucleic acid. In some embodiments, the proportion of local anesthetic to nucleic acid in the vesicles is 0.05-1.25% (w/v) local anesthetic to 100 xcexcg/ml-l mg/ml nucleic acid. In some embodiments, the proportion of local anesthetic to nucleic acid in the vesicles is 0.1-0.5% (w/v) local anesthetic to 500 xcexcg/ml-1500 xcexcg/ml nucleic acid. In some embodiments, the lamellar vesicles up to about 200 nm diameter as measured by scanning electron microscopy. In some embodiments, the lamellar vesicles are 50-150 nm diameter as measured by scanning electron microscopy. In some embodiments, greater than 50% of said lamellar vesicles greater that 300 nm diameter as measured by scanning electron microscopy. In some embodiments, greater than 50% of said lamellar vesicles are less than 400 nm diameter as measured by scanning electron microscopy. In some embodiments, the local anesthetic is bupivacaine, etidocaine, tetracaine, procainamide, chloroprocaine, ropivacaine, prylocaine, mepivacaine, lidocaine, procaine, carbocaine, methyl bupivacaine or cocaine. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is plasmid DNA. In some embodiments, the lamellar vesicles further comprise one or more compounds selected from the group consisting of: cationic lipids, neutral lipids, anionic lipids, cationic surfactants, neutral surfactants, anionic surfactants, cationic detergents, neutral detergents, and anionic detergents. In some embodiments, the lamellar vesicles further comprise one or more compounds selected from the group consisting of: proteins, polypeptides, peptides, and non-proteinaceous drugsltherapeutic compounds. In some embodiments, the nucleic acid is DNA comprising a DNA sequence that encodes an immunogen.
The present invention further relates to methods of preparing compositions that comprise lamellar vesicles. The methods comprise the step of forming the lamellar vesicles by combining 0.01-2.5% (w/v) local anesthetic with 1 xcexcg/ml-10 mg/ml nucleic acid in the presence of 0-2M salt at a pH of 4-8.5. In some embodiments, the lamellar vesicles are formed by combining 0.05-1.25% (w/v) local anesthetic with 100 xcexcg/ml-1mg/ml nucleic acid in the presence of 0-500 mM salt at a pH of 6-7.5. In some embodiments, the lamellar vesicles are formed by combining 0.1-0.5% (w/v) local anesthetic with 500 xcexcg/ml-1500 xcexcg/ml nucleic acid in the presence of 0-500 mM salt at a pH of 6-7.5. In some embodiments, the lamellar vesicles are formed by combining 0.1-0.5% (w/v) local anesthetic with 500 xcexcg/ml-1500 xcexcg/ml nucleic acid in the presence of greater than 2M salt at a pH of 6-7.5 followed by removal of salt to below 500 mM. In some embodiments, proteins, polypeptides, peptides, or non-proteinaceous drugs/therapeutic compounds are also combined with the local anesthetic and nucleic acid to produce lamellar vesicles that include the proteins, polypeptides, peptides, or non-proteinaceous drugs/therapeutic compounds.
The present invention further relates to a method of delivering a protein, which includes polypeptides and peptides, to a cell of an individual. The method comprises the step of administering to the individual a composition that comprises lamellar vesicles that comprise a local anesthetic and a nucleic acid molecule. The lamellar vesicles comprise nucleic acid molecules that comprise a nucleotide sequence that encodes the protein operably linked to regulatory sequences. The nucleic acid molecules are taken up by cells of the individual and expressed to produce the protein.
The present invention further relates to a method of delivering a protein, polypeptide, peptide, non-proteinaceous drug/therapeutic compound, intact viral particle or fragment thereof, or microorganism to a cell of an individual. The method comprises the step of administering to the individual a composition that comprises lamellar vesicles that comprise a local anesthetic, a nucleic acid molecule and the protein, polypeptide, peptide, or non-proteinaceous drug/therapeutic compound.
The present invention further relates to a method of inducing an immune response in an individual. The method comprises the step of administering to the individual a composition that comprises lamellar vesicles that comprise a local anesthetic and a nucleic acid molecule. The lamellar vesicles comprise nucleic acid molecules that comprise a nucleotide sequence that encodes an immunogen operably linked to regulatory sequences. The nucleic acid molecules are taken up by cells of the individual, expressed to produce the immunogen and an immune response is generated against the immunogen by the individual. In some embodiments, the lamellar vesicles further comprise an immunogenic protein or peptide. In some embodiments, lamellar vesicles further comprise a physiologically active protein.
The present invention further relates to a method of delivering a nucleic acid molecule to a cell of an individual. The method comprises the step of administering to the individual a composition that comprises lamellar vesicles that comprise a local anesthetic and a nucleic acid molecule. The lamellar vesicles comprise nucleic acid molecules that comprises a nucleotide sequence that encodes said protein operably linked to regulatory sequences, an antisense oligonucleotide, a ribozyme or a triplex forming nucleic acid molecule.